1. Field of the Invention
The present invention relates to cell biology, and more particularly to characterizing a cell by its nuclear packing efficiency (NPE).
2. Background Information
Virtually all eucaryotic cells have a nucleus, a compartment enclosed by the nuclear membrane and containing most of the cell""s DNA, as well as other nuclear components. Aberrations in the size and shape of the nucleus have long been recognized as an indication of cancer and other diseases, although their characterization was previously limited to microscopic observation.
Since then, attempts have been made to characterize cells by quantitating and comparing various nuclear components. For example, measurements of DNA, RNA and nuclear protein have been compared with each other. One study measured the quantity of DNA and tried to correlate it with measurements of the size of the nucleus, as measured by light scatter, time of flight and area. However, these correlations have been hampered by unreliable indirect estimates of the nuclear volume.
Indirect estimates of volume from diameter measurements using light scatter have worked best assuming uniform spherical particles of a certain size range and having a relatively high index of refraction. But this technique becomes less accurate when applied to nonideal biological samples outside the optimal range of measurement. Other measurements, termed xe2x80x9ctime-of-flightxe2x80x9d or TOF, measure the size of particles as a flowstream carries the particles across a beam of light. However, this technique is subject to many limitations, including sensitivity to fluctuations in the speed of the particles and variations from the relative orientation of the particles, and only yields a measurement of one axis of the three-dimensional particle.
Still other indirect measurements estimate nuclear volume based on the cross-sectional areas of the nucleus. But these measurements, in turn, can be limited by variability in the staining and mounting techniques used on the nuclei. In particular, confocal microscopes have been used to measure the area of stained DNA in the nucleus, summing up successive cross-sections to obtain a measure of the total DNA. By assuming that the nuclear volume is proportional to the stained DNA, this technique then yields an estimate of the nuclear volume. However, this technique fails to account for the granularity of DNA within in the nucleus, and ignores the varying contribution to nuclear volume from the other components of the nucleus: RNA, nuclear proteins, nuclear lipids, nuclear envelope and nuclear water.
Thus, previous techniques fail to meet the need for satisfactory measurements of nuclear volume in combination with a useful correlation with other measurements to characterize the condition of nuclei and the cells as a whole. The present invention satisfies this need and provides related advantages as well.
The nucleus of a cell is a highly organized structure, allowing precursor materials to pass through pores in the nuclear envelope into the nucleus, and nuclear products to be transported out to the cytoplasm. The complex nuclear machineryxe2x80x94nucleic acids, proteins, lipids and other componentsxe2x80x94are tightly packaged within the volume of the nucleus.
As with most organized structures in nature, the nucleus assumes a shape for compact packaging of its components for optimal efficiency. When a cell becomes diseased, such as in malignant cells, the nuclear organization breaks down. For example, the DNA loses its ability to fold efficiently around histone proteins into organized structures called nucleosomes. The protein content also changes, as well as other biochemical components of the nucleus. The volume of the nucleus becomes forced to increase to accommodate this disorganization. Thus, the efficiency of this packing is a characteristic of the nucleusxe2x80x94and a useful indication of the condition of the cell as a whole.
The present invention provides methods and devices for determining the nuclear packing efficiency (NPE) of a cell by measuring the spatial displacement of the nucleus (SDN), for example by using flow cytometry to measure electronic nuclear volume (ENV). When the method is applied to procaryotes or viruses, the SDN can be considered the volume of the surrounding particle, which is the procaryotic cell or the virus itself. One or more biochemical components (BCs) of the nucleus are also measured, such as nucleic acids, nuclear protein, nuclear lipids or nuclear water. An NPE is then determined by correlating the values measured for BC and SDN.
A variety of techniques can be used to correlate the BC and SDN to yield an NPE. Polynomial fitting can be used, from the ratio BC/SDN to more complex expressions such as NPE=k1(BC)a/(SDN)b+k2(BC)c+k3(SDN)d+k4. Graphical methods are particularly useful for evaluating NPEs for a population of cells and for identifying distinct subpopulations of cells. Subpopulations can then be characterized in terms of their geometric parameters, such as diameter, eccentricity and gradient line slope.
Once determined, NPEs are useful for identifying cells having a phenotype of interest. For example, cells can be identified by tissue source and by the sex and species of the organism, as well as by various states of differentiation and stages in the cell division cycle and apoptosis. NPEs can also identify cells having various disease states as they differ from their normal states, particularly neoplastic cells exhibiting aneuploidy, distinguishing among benign, malignant and metastatic cells, thus enabling the diagnosis and prognosis of cancer.